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Genes (Basel). 2017 Oct 11;8(10). pii: E265. doi: 10.3390/genes8100265.

Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.).

Author information

1
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. yuyongting@caas.cn.
2
College of Pharmacy and Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs Research, Shaanxi University of Chinese Medicine, Xi'an 712406, China. jay_gumling2003@aliyun.com.
3
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Lizhimin@caas.cn.
4
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Chengyi@caas.cn.
5
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Csgao06@163.com.
6
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Zengliangbin@caas.cn.
7
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Chenjia01@caas.cn.
8
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Yanli214@126.com.
9
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. Sunxp66@163.com.
10
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. guolitao@caas.cn.
11
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China. yanzhun71@gmail.com.

Abstract

Phytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie (Boehmeria nivea L.) phytocystatin gene, designated as BnCPI, was isolated from a ramie cDNA library and its full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence (691 bp) consisted of a 303 bp open reading frame (ORF) encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical isoelectric point (pI) of 6.0. The alignment of genome DNA (accession no. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly⁵-Gly⁶ at the N-terminal, the reactive site motif QxVxG (Q49V50V51S52G53), the L79-W80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L22G23R24 F25A26V27 D28D29H30 N31) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). The BnCPI was subcloned into expression vector pSmart-I and then overexpressed in Escherichia coli BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (Nα-benzoyl-L-arginine-2-naphthyamide), as well as the mycelium growth of some important plant pathogenic fungi. The data further contribute to our understanding of the molecular functions of BnCPI.

KEYWORDS:

BnCPI; Boehmeria nivea L.; cysteine protease inhibitors; functional expression; intron

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